Regulation of mRNA processing: Mechanisms and Consequences

 描述（由申请人提供）：真核细胞中mRNA的合成是一个高度复杂的过程，除了转录本身之外，还包括mRNA前体的剪接和3'端形成。我的实验室多年来一直在研究这些过程的机制和调节，并展示了它们如何与其他细胞事件（如DNA损伤）整合，以及它们如何在分化和疾病中发挥作用。这个建议结合了我们在这些领域的研究，可以分为以下三大领域：1）mRNA加工和疾病。将继续研究与癌症和神经退行性疾病中RNA代谢有关的几种蛋白质的作用。RNA加工因子在癌症中的作用反映了两种不同的机制，剪接调节蛋白的过表达和编码各种剪接因子的基因突变。关于前者，将继续进行源于阐明癌症中PKM选择性剪接（AS）失调机制的研究的正在进行的实验。重要的问题包括机制的一般性，剪接因子过表达对肿瘤发展的重要性，以及关键的调节剪接事件的身份。剪接因子突变可引起MDS和某些白血病，并且正在进行的研究旨在阐明突变影响几种基因功能的机制，例如，SRSF 2和SF 3B 1，以及这些缺陷如何导致疾病。关于神经退行性疾病，工作集中在如何ALS引起突变的RNA/DNA结合蛋白TLS/FUS破坏蛋白质功能;如何在C9 ORF 72的六核苷酸重复扩增形成G-四链体结构，隔离hnRNP H，破坏AS，并有助于ALS;以及如何在RNA/DNA解旋酶Senataxin致病突变影响其在预防转录诱导的DNA损伤的作用。2)mRNA加工和分化。研究如何在AS和替代多聚腺苷酸化（阿帕）的变化有助于人类胚胎干细胞的多能性和分化将继续进行。AS如何影响转录调节因子TCF 3的功能以及这如何有助于分化将被确定。AS事件的机制，包括剪接调节器本身是如何调节的，将进行调查。我们意想不到的发现，编码两个PA因子的转录本的AS，这两个PA因子都是CPSF的亚基，并且与AAUAAA识别有关，在分化过程中发生了改变。不同的异构体如何影响阿帕和分化，将被确定，因为它们影响PA位点选择的机制。3)RNA加工和转录。将继续进行检查RNA pol II CTD（由26-52个七肽重复序列组成的独特结构域（共有YSPTSPS））功能的实验。例如，最近的实验确定了P-Tyr 1残基在促进某些lncRNA周转中的作用，并表明 这涉及与RNA解旋酶Mtr 4和PA机制的相互作用。这些和其他实验将促进我们对连接转录和RNA加工的“CTD代码”的理解。

 DESCRIPTION (provided by applicant): Synthesis of mRNAs in eukaryotic cells is a highly complex process, including, in addition to transcription itself, splicing and 3' end formation of mRNA precursors. My laboratory has studied the mechanisms and regulation of these processes for many years, and also shown how they are integrated with other cellular events such as DNA damage, and also how they function in differentiation and disease. This proposal combines our studies in these areas, and can be divided into the following three broad areas: 1) mRNA processing and disease. Studies examining the roles of several proteins implicated in RNA metabolism in cancer and neurodegenerative disease will be pursued. The role of RNA processing factors in cancer reflects two distinct mechanisms, overexpression of splicing regulatory proteins and mutation of genes encoding various splicing factors. With respect to the former, ongoing experiments stemming from studies elucidating the mechanism underlying deregulation of PKM alternative splicing (AS) in cancer will be pursued. Important questions include the generality of the mechanism, the importance of splicing factor overexpression to tumor development, and the identity of the critical regulated splicing events. Splicing factor mutations can cause MDS and certain leukemias, and ongoing studies are aimed at elucidating mechanisms by which mutations affect the function of several, e.g., SRSF2 and SF3B1, and how such defects lead to disease. With respect to neurodegenerative disease, work centers on how ALS-causing mutations in the RNA/DNA-binding protein TLS/FUS disrupt protein function; how the hexanucleotide repeat expansions in C9ORF72 form G-quadruplex structures that sequester hnRNP H, disrupt AS, and contribute to ALS; and how disease-causing mutations in the RNA/DNA helicase Senataxin affect its role in prevention of transcription-induced DNA damage. 2) mRNA processing and differentiation. Studies examining how changes in AS and alternative polyadenylation (APA) contribute to human embryonic stem cell pluripotency and differentiation will be pursued. How AS affects the function of the transcriptional regulator TCF3 and how this contributes to differentiation will be determined. The mechanism of the AS event, including how the splicing regulators are themselves regulated, will be investigated. Our unexpected finding that AS of transcripts encoding two PA factors, both subunits of CPSF and implicated in AAUAAA recognition, is altered during differentiation will be pursued. How the distinct isoforms affect APA, and differentiation, will be determined, as will the mechanism by which they influence PA site choice. 3) RNA processing and transcription. Experiments examining the function of the RNA pol II CTD, a unique domain consisting of 26-52 heptad repeats (consensus YSPTSPS), will be continued. For example, recent experiments establishing a role for P-Tyr1 residues in facilitating turnover of certain lncRNAs, and suggesting that this involves interaction with the RNA helicase Mtr4 and the PA machinery, will be pursued. These and other experiments will advance our understanding of the "CTD code" that links transcription and RNA processing.